摘要
含有猪细小病毒SC1株非结构蛋白NS1基因的重组质粒pPNS1通过EcoR+Hind双酶切后,回收NS1基因,将其亚克隆进原核表达载体pET30a(+)中,构建了重组表达质粒pET-NS1,转入表达宿主菌BL21中,通过IPTG的诱导表达,SDS-PAGE电泳结果表明,重组NS1蛋白高效表达,以包涵体形式存在,占细菌总蛋白的26.7%。并确定了NS1蛋白的最佳诱导条件:IPTG终浓度为0.6mmol/L,诱导时间为3h。Westernblotting检测表明,表达产物具有良好的免疫原性。重组蛋白经纯化后作为抗原,包被酶标板,以辣根过氧化物酶标记葡萄球菌A蛋白作为二抗,建立了辣根过氧化物酶标记葡萄球菌A蛋白的酶联免疫吸附试验(PPA-NS1-ELISA)。结果表明,抗原最佳包被质量浓度为31.3mg/L,血清最佳稀释度为1∶40。阳性标准初步定为:待检血清D490>0.32,且待检血清D490/阴性血清D490>2.0。
The recombinant plasmid pPNS1 was digested with EcoR I and Hind it to generate NS1 gene, the fragment was then cloned into pET30a （＋） to get a prokaryotic plasmid pET-NS1 ; The target gene was expressed in very high level as inclusion body in the host cell BL21（DE3） when induced with IPTG. The expression was opti mized with proper inducing conditions of 0. 6 mmol/L IPTG for 3 hours induction. The highest expression of the target protein amounts to 26.7% of the total bacterial protein. Western blotting analysis proved the recombinant protein has a good reactive ability against PPV positive serum, Horseradish peroxidase-staplylococcal protein （HRP-SPA） was used as the second antibody and PPA-NS1-ELISA was developed in the experiment. The optional working circumstances for the PPA-NS1-ELISA assay including the optimal coated antigen concentration of 31.3 mg/L and the optimal serum dilution of 1 ： 40. The positive criterion of PPA-NS1-ELISA assay is the tested serum D490〉0.32 and the tested serum D450/the negtive serum D490〉2.0.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2007年第1期13-16,共4页
Chinese Journal of Veterinary Science
基金
四川省"十五"攻关项目
四川省科技厅应用基础资助项目(03JY029-035-2)
